Many pathogenic bacteria must compete with the mammalian host for the essential metal iron, even as the host sequesters circulating iron and releases reactive oxygen intermediates to damage bacterial metalloenzymes as its first line of defense. Iron-sulfur (Fe-S) cluster cofactor biogenesis is critical to the survival of bacterial pathogens. A concerted multi-protein system encoded by the suf operon provides bacteria with a protected Fe-S biogenesis pathway under these conditions, which are detrimental to thiol and iron chemistry. The suf operon, conserved in Escherichia coli and in pathogenic Mycobacterium tuberculosis, encodes six proteins whose functions are still being elucidated. The SufS cysteine desulfurase requires the SufE sulfur transfer partner protein as well as the SufBC2D scaffold complex for full activity during sulfur trafficking. Sulfur donation from SufE to the SufB scaffold protein requires that SufB also be in a complex with SufC. The SufC ATPase is dependent on interactions with SufB and/or SufD for activation of the ATP hydrolysis cycle. In vivo Fe-S cluster assembly on SufB also requires the SufD protein. Finally, the SufA Fe-S cluster trafficking protein preferentially interacts with the [4Fe-4S] form of SufBC2D rather than the apo form of the scaffold complex. Thus Suf Fe-S cluster assembly is synchronized by a complicated network of protein- protein interactions (PPIs). The tight control of SufS and SufC enzyme activity and the careful regulation of sulfur and Fe-S cluster trafficking are critical for Suf funtion during iron starvation and oxidative stress in vivo. Our long-term goal is to characterize PPIs critical for iron metallocofactor biogenesis in order to disrupt those interactions with novel, rationally designed antibiotics. The hypothesis being tested in this proposal is that PPIs and their coupled conformational responses coordinate the steps of Suf stress-responsive Fe-S cluster biogenesis. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) will be combined with biophysical, biochemical, and genetic approaches to test this hypothesis. The specific aims of the proposal are to (1) define the full mechanism of sulfur transfer from SufS to SufE to SufB scaffold complexes, (2) characterize the conformational dynamics of the SufBC2D Fe-S cluster scaffold complex, and (3) determine the mechanism of Fe-S cluster transfer from SufB to the Fe-S carrier SufA. Completion of the experiments in this proposal will provide structural insight into the mechanistic roles of PPIs during Fe-S biogenesis by the Suf pathway. This insight will allow us to target key PPIs in the Suf pathway using novel antibiotic strategies.